Antibiotics, neoviridogriseins, and their method of production

ABSTRACT

A group of novel depsipeptide antibiotics, neoviridogriseins I, II and III are produced by fermentation of Streptomyces sp. P8648 (FERM-P 3562; ATCC 31289). The antibiotics are highly active against Gram-positive bacteria and mycoplasmas.

CROSS-REFERENCE TO RELATED APPLICATION

This is a divisional of application Ser. No. 333,950, filed Dec. 23,1981, now U.S. Pat. No. 4,456,592, which is a divisional of Ser. No.803,922, filed June 6, 1977, now U.S. Pat. No. 4,355,112.

BACKGROUND OF THE INVENTION

(1) FIELD OF THE INVENTION

This invention relates to new depsipeptide antibiotics, calledneoviridogriseins I, II and III; to their production and isolation; andto their utilization as therapeutic drugs and animal feed additive.

(2) DESCRIPTION OF THE PRIOR ART

The mikamycin-vernamycin (or streptogramin) family of antibiotics areclassified into two major groups. One major group (Group A according toD. Vazquez; pp521-534, ANTIBIOTICS III, Mechanism of action ofantimicrobial and antitumor agents, Springer-Verlag Berlin HeidelbergNew York 1975) is a macrocyclic lactone compound, and includesgriseoviridin, ostreogrycin A(=mikamycin A=virginiamycin M=staphylomycinM=pristinamycin IIA=vernamycin A=streptogramin A=synergistinA-1=Compound PA-114A1=Compound E-129 Factor A, etc.), ostreogrycin G(=virginiamycin MII=staphylomycin MII=pristinamycinIIB=dihydrostreogrycin A=Compound E-129 Factor B=Compound R.P.-13920,etc.), and Compounds A-2315 A, B and C. Though bacteriostatic, thisgroup of antibiotics are active against Gram-positive bacteria andmycoplasmas. Griseoviridin, which is one of the fermentation products inthis invention, is known to be produced together with viridogrisein bystreptomycetes, and the production of griseoviridin is disclosed in U.S.Pat. Nos. 3,023,204 and 3,174,902.

The other major group (Group B according to D. Vazquez) is a macrocyclicdepsipeptide compound and divided into two sub-groups, that is, theviridogrisein sub-group and the vernamycin B sub-group. The antibioticsin this major group are mainly effective in suppressing the growth ofGram-positive bacteria such as Staphylococcus aureus and Bacillussubtilis. The vernamycin B sub-group contains 12 homologues. Because thesynonym relationships among the published names of antibiotics in thisgroup are so complicated, it is practically impossible to list all thecompound names of this sub-group. Some representative, andscientifically important, compounds of this sub-group are as follows:

1. Vernamycin B=ostreogrycin B=pristinamycin IA=mikamycinB=streptogramin B=synergistin B-1=compound E-129 Factor Z=compoundPA114B1

2. Vernamycin B=ostreogrycin B1=pristinamycin IC=compound E-129 FactorZ1

3. Vernamycin B=ostreogrycin B2=pristinamycin IB=compound E-129 FactorZ2=compound R.P.-13919

4. Ostreogrycin B3=compound E-129 Factor Z3

5. Patricin A

6. Patricin B

7. Vernamycin B.sub.δ

8. Vernamycin C=doricin

9. Virginiamycin (Staphylomycin)S

10. Virginiamycin(Staphylomycin)S2

11. Virginiamycin (Staphylomycin)S3

12. Virginiamycin (Staphylomycin)S4 (or S1)

The compounds of this sub-group have the common feature that they arecomposed of seven constituents sharing in the same four constituents of3-hydroxypicolinic acid, L-threonine, L-proline and L-phenylglycine. Onthe other hand, in the viridogrisein sub-group there has been known onlyone compound, viridogrisein, and the identity with viridogrisein ofetamycin, compound K-179 and compound F-1370A is well established. Incontrast to the above vernamycin B sub-group, viridogrisein is composedof 8 constituents, that is, 3-hydroxypicolinic acid, L-threonine,D-leucine, 4-hydroxy-D-proline, sarcosine, β,N-dimethyl-L-leucine,L-alanine and L-phenylsarcosine. As apparent in the following detaileddescription of the invention, the neoviridogrisein antibiotics of thisinvention belong to this viridogrisein sub-group, and one of thecompounds, neoviridogrisein IV, has been identified as viridogrisein.Preparation of viridogrisein is disclosed in U.S. Pat. No. 3,023,204.

The synergism between Group A and Group B is well known on variousmicroorganisms, and profitably utilized to potentiate the therapeuticefficiency of pharmaceutical drugs containing the antibiotic of thisfamily as active components. This is true of neoviridogriseins, too.

SUMMARY OF THE INVENTION

This invention relates to novel and useful depsipeptide antibioticsubstances. More particularly, it relates to a group of depsipeptideantibiotics called neoviridogriseins I, II and III and viridogrisein.They are active against Gram-positive bacteria and mycoplasmas, and canbe used in diluted forms, as crude concentrates, or in pure form,together with or without griseoviridin.

It is an object of the present invention to provide a group of newdepsipeptide antibiotics called neoviridogriseins I, II and III whichare highly effective in inhibiting the growth of various Gram-positivebacteria and mycoplasmas.

Another object of this invention is to give a method for the preparationof these depsipeptide antibiotics by fermenting a suitable aqueousmedium under aerobic conditions by a new species of Streptomyces namedStreptomyces sp. P8648 (FERM-P3562; ATCC31289) whereby formedneoviridogriseins are recovered from the fermentation broth togetherwith or without griseoviridin.

It also is another object of the present invention to provide a methodfor the selective production of the desired neoviridogriseincomponent(s) by supplementing some pertinent amino acid(s), moreparticularly, proline to increase neoviridogriseins I and II, andalpha-amino-n-butyric acid to increase neoviridogriseins I and III,during or before fermentation. Other objects will be apparent from thedetailed description of the invention. Neoviridogriseins I, II and IIIof the present invention are valuable antibiotics which are highlyactive against various pathogenic microorganisms including Gram-positivebacteria and mycoplasma, and, accordingly, find utility in human andveterinary medicine. More practically, the antibiotics of this inventioncan be used as therapeutic drugs for the treatment of infectionsdiseases caused by Gram-positive bacteria and mycoplasma, for example,Staphylococcus aureus, Streptococcus pyogenes, Diplococcus pneumoniae,Mycoplasma gallisepticum, Mycoplasma fermentans, Mycoplasma agalactiae,etc.

In this invention, the unmodified term "neoviridogrisein" is used togenerically designate depsipeptide antibiotics called neoviridogriseins;that is, it means not only a member in the group of neoviridogriseins I,II and III and viridogrisein(=neoviridogrisein IV), but also a mixtureof any two or more members selected from the said group.

DESCRIPTION OF THE DRAWINGS

FIG. 1: UV spectrum of neoviridogrisein I (NVG I) in methanol

FIG. 2: UV spectrum of neoviridogrisein II (NVG II) in methanol

FIG. 3: UV spectrum of neoviridogrisein III (NVG III) in methanol

FIG. 4: UV spectrum of neoviridogrisein IV (NVG IV, VG) in methanol

FIG. 5: UV spectrum of neoviridogrisein I (NVG I) in methanol/NaOH 0.1N

FIG. 6: UV spectrum of neoviridogrisein II(NVG II) in methanol/NaOH 0.1N

FIG. 7: UV spectrum of neoviridogrisein III(NVG III) in methanol/NaOH0.1N

FIG. 8: UV spectrum of neoviridogrisein IV (NVG IV,VG) in methanol/NaOH0.1N

FIG. 9: I.R. spectrum of neoviridogrisein I (NVG I)-KBr tablet

FIG. 10: I.R. spectrum of neoviridogrisein II (NVG II)-KBr tablet

FIG. 11: I.R. spectrum of neoviridogrisein III (NVG III)-KBr tablet

FIG. 12: I.R. spectrum of neoviridogrisein IV (NVG IV,VG)-KBr tablet

DETAILED DESCRIPTION OF THE INVENTION MICROORGANISM

The new depsipeptide antibiotics of the invention are produced by a newstrain of Streptomyces called Streptomyces sp. P8648 (FERM-P 3562; ATCC31289) together with viridogrisein and griseoviridin. This microorganismhas been isolated from a soil sample collected near the Kuzuryu dam inFukui-ken.

Generally, the microorganism of this invention elongates colorless,short aerial mycelia from well-branched (single branching) substratemycelia. Spore chains with smooth surface are formed in a loose loop ontop of aerial mycelia. Neither whirl nor ascospore is observed. Thecultural characteristics of this microorganism on various agar media aredescribed as follows:

(1) Sucrose-nitrate agar

Growth: Poor

Aerial mycelium: Thin, white aerial mycelia occasionally formed

Reverse: Colorless to grayish white.

Soluble pigment: None.

(2) Glucose-asparagine agar

Growth: Abundant

Aerial mycelium: Little or none. When formed, white.

Reverse: Pale yellowish white to light yellow.

Soluble pigment: None.

(3) Glycerin-asparagine agar

Growth: Moderate

Aerial mycelium: Little or none. When formed, white.

Reverse: Pale yellow to grayish yellow

Soluble pigment: None.

(4) Yeast extract-malt extract agar

Growth: Abundant

Aerial mycelium: White to white with grayish tinge.

Reverse: Light yellow, later turning to brownish gray

Soluble pigment: None or rarely slight brown.

(5) Starch agar

Growth: Moderate

Aerial mycelium: None or little. When formed, white.

Reverse: Pale yellow with light grayish tan in the center of colonies

Soluble pigment: None.

Starch hydrolysis: Poor.

(6) Tyrosine agar

Growth: Moderate

Aerial mycelium: None or a few spots of white aerial myceliumoccasionally observed.

Reverse: Grayish yellow to light yellowish tan

Soluble pigment: Initially pale purple to light reddish brown, 10 dayslater turning to pale brown. Little melanoid pigment formed.

(7) Nutrient agar

Growth: Good.

Aerial mycelium: Thin, white.

Reverse: Pale yellow

Soluble pigment: None.

(8) Oatmeal agar

Growth: Good.

Aerial mycelium: White to grayish white.

Reverse: Grayish yellow to light reddish brown with grayish tinge

Soluble pigment: None.

The optimum growth temperature for the microorganism of this inventionis in the range of 25°-35° C. Though the growth is very poor, themicrobe can grow even at a temperature beyond the said temperature rangelike 10° C. or 45° C. But it cannot grow at a temperature of 52° C. Thisactinomycete liquefies gelatin in glucose-peptone-gelatin medium; weaklyhydrolyzes starch in starch-inorganic salts agar; and peptonizes skimmedmilk without coagulation.

Production of melanoid pigment is occasionally observed in tyrosineagar, but not in peptone-yeast extract-iron agar and tyrosine-yeastextract broth.

Carbon source assimilation pattern of this microorganism is as follows(In Pridham-Gottlieb's medium):

Positive: D-Xylose, D-glucose, D-fructose L-rhamnose,D-mannitol.

Slightly positive: Sucrose

Negative: L-Arabinose, i-inositol, raffinose.

In relation to the production of the known peptide and non-peptidemacrolide antibiotics like mikamycins A and B, virginiamycins,ostreogrycins, etamycin, vernamycins, viridogrisein, griseoviridin andpristinamycins, the following microorganisms should be compared withStreptomyces sp. P8648:

    ______________________________________                                               Streptomyces griseus NRRL 2426                                                griseoviridus NRRL 2427                                                       sp., etamycin producer                                                        conganensis                                                                   ostreogriseus                                                                 mitakaensis                                                                   loidensis                                                              ______________________________________                                    

The available information on the cultural and physiologicalcharacteristics of the said microorganisms shows clear differencesbetween the streptomycete claimed in this invention and theabove-mentioned ones. For example, Streptomyces griseus NRRL 2426differs in that it belongs to the Section Rectiflexibiles with straightor slightly wavy spore chains while the microorganism of this inventionis included in the Section Spirales; that the former produces gray toyellowish gray aerial mycelia on yeast extract-malt extract agar whilethe latter produces white to grayish white aerial mycelia; and that theformer utilizes L-arabinose while the latter does not. Streptomyces sp.,etamycin producer which has been specified in Antibiotics Annual1954-1955, pp. 728-732, can be differentiated from the microorganism ofthis invention in the assimilation pattern of carbon sources and thecultural characteristics on Czapek agar, glucose-asparagine agar andnutrient agar. Streptomyces conganensis shows clear differences in themorphological characteristics of spores. Among the above-listedmicroorganisms, Streptomyces griseoviridus NRRL 2427 looks most similarto the streptomycetes of this invention. The results of the taxonomicalcomparison between the said two type cultures are summarized in thefollowing table:

    ______________________________________                                        Streptomyces                                                                  griseoyiridus       Streptomyces                                              NRRL 2427           sp. P 8648                                                ______________________________________                                        Color of                                                                              Pale orangish yellow                                                                          White to grayish white                                aerial  to yellowish pink                                                                             aerial mycelium poorly                                mycelium                                                                              with gray tinge on                                                                            formed on most ISP media.                                     yeast extract-malt                                                                            White aerial mycelium                                         extract agar, oatmeal                                                                         abundantly formed on                                          agar, starch agar and                                                                         yeast extract-malt                                            glycerin-asparagine                                                                           extract agar.                                                 agar.                                                                 Color or                                                                              Grayish yellow to                                                                             Pale yellow or light                                  substrate                                                                             olivish brown or                                                                              yellow to grayish                                     mycelium                                                                              blackish brown on                                                                             brown on most ISP                                             yeast extract-malt                                                                            media.                                                        extract agar, oatmeal                                                         agar, starch agar and                                                         glycerin-asparagine                                                           agar.                                                                 Soluble No melanoid pigment                                                                           No melanoid pigment                                   pigment formed. No other                                                                              formed. No other pigment                                      pigment usually usually observed, but                                         observed, but rarely                                                                          rarely brown pigment                                          yellow pigment poorly                                                                         slightly formed.                                              formed.                                                               Utilization                                                                           L-arabinone +++ L-arabinose -                                         of carbon                                                                             D-fructose ± D-fructose +++                                        sources sucrose -       sucrose ±                                          ______________________________________                                    

As apparent from the preceding table, clear differences have beenconfirmed between Streptomyces griseoviridus NRRL 2427 and thestreptomycete of this invention in the morphological and culturalcharacteristics and the utilization pattern of carbon sources. Inaddition, when both are fermented under identical conditions, themicroorganism of this invention can produce neoviridogriseins I, II andIII as well as viridogrisein(neoviridogrisein IV) and griseoviridin,while the type culture of Streptomyces griseoviridus NRRL 2427 producesonly viridogrisein and griseoviridin, but not neoviridogriseins I, IIand III.

From the above described results, the microorganism employed in thisinvention has been concluded to be a new species of Streptomyces andnamed Streptomyces sp. P 8648. The type culture of this microorganismhas been deposited with the Fermentation Research Institute, Agency ofIndustrial Science and Technology, with the deposition number of FERM-PNo. 3562. It has also been deposited with the American Type CultureCollection where it was assigned the code number ATCC 31289.

It may be well understood to those skilled in the art that thisinvention is not limited to the particular microorganism which has beenspecified above and filed as FERM-P No. 3562 to Fermentation ResearchInstitute, but includes all those spontaneous and artificial mutantsderived from the said microorganism which are capable of producing thenew antibiotics, neoviridogrisein I, II and III.

PRODUCTION OF NEOVIRIDOGRISEINS I, II, AND III

The antibiotics of this invention are produced by inoculating andpropagating Streptomyces sp. P8648 in a suitable medium under aerobicconditions at a temperature in the range of 18°-37° C. for a period of2-14 days whereby the accumulated antibiotics are recovered from thefermentation broth and purified by conventional methods. The preferredembodiments of the processes according to this invention will beillustrated in more detail in the following:

For the fermentation of the microorganism of this invention, employableare all kinds of media which have been well known as media forStreptomycetes. For example, preferable carbon sources of the medium areglucose, glycerin, starch, dextrin, oatmeal, molasses, fat and oil andthe like. Suitable nitrogen sources for the purposes of this inventionare: soybean meal, cotton seed meal, meat extract, peptone, dry yeast,corn steep liquor, yeast extract, casein and its hydrolysate andinorganic salts such as ammonium sulfate and ammonium nitrate. Ifdesired, minor growth factors may be added to the medium. They includevitamins, amino acids, organic and inorganic salts such as calciumcarbonate, sodium chloride, potassium chloride, sodium phosphate,potassium phosphate and magnesium sulfate.

The new antibiotics of this invention can be produced by fermentation intraditional vessels such as a shake flask, a jar fermentor and a tankfermentor, but economically the submerged cultivation under forcedaeration will be most advantageous in an industrial scale. Thefermentation is desirably carried out under aerobic conditions at atemperature in the range of 25°-35° C. When a shake flask or a tankfermenter is employed, the production of neoviridogriseins reaches apeak usually in 2-10 days. The pH value during fermentation may changebeyond the physiological range, depending on the kind of mediumemployed. It is more desirable to adjust and maintain the pH valueduring fermentation in the range of 6-9. Usually the pH of the medium isadjusted to 6.5-8.5 before inoculation.

CONTROL OF THE NEOVIRIDOGRISEIN COMPONENTS IN THE FERMENTATION BROTH

As described above, the microorganism of this invention produces amixture of neoviridogriseins I, II, III and IV and griseoviridin. It ispossible to change the composition of neoviridogriseins in thefermentation broth by a suitable combination of carbon and nitrogensources in the medium without the specific addition of either a freeamino acid or an organic acid. But it will be more profitable from theviewpoint of industrial production to adjust the content ofneoviridogriseins I, II and/or III in the fermentation broth by addingto the medium the pertinent constituent amino acid(s) in free formduring fermentation, depending on the circumstances and the demand. Thecomposition of neoviridogriseins in the fermentation broth may be variedappropriately by selecting spontaneous or artificial mutants derivedfrom the type culture of the streptomycete of this invention; byadjusting fermentation conditions like temperature, pH and aeration;and/or by adding to the medium physiologically active agents such asenzyme inhibitors and promoters. One of the preferred embodiments ofmethods for selective production of particular neoviridogriseincomponents consists of feeding the pertinent constituent amino acid(s),alpha-amino-n-butyric acid and/or proline during fermentation. Moreparticularly, the addition of proline during fermentation increases thepercentage of neoviridogriseins I and II in the total amount ofneoviridogriseins I, II, III and IV, which are more potent inantimicrobial activities than neoviridogriseins III and IV. This is alsotrue of alpha-amino-n-butyric acid. The amount of neoviridogriseins Iand III can be selectively increased by feeding alpha-amino-n-butyricacid to the medium before inoculation or during fermentation. As themicroorganism of this invention produces protease during growth,proteinaceus material which contains the said pertinent constituentamino acid(s) may be added instead of the free amino acid(s). Forexample, proline can be substituted by casein or correspondinghydrolysates from acid hydrolysis such as, for instance, casamino acids.

ISOLATION AND PURIFICATION

The new antibiotics, neoviridogriseins I, II and III and viridogriseincan be isolated from the fermentation broth by conventional methodsbased on their physico-chemical properties as depsipeptide antibiotics.If necessary, neoviridogriseins may be recovered from the fermentationbroth together with griseoviridin as a neoviridogriseins-griseoviridinmixture. When they are prepared for feed additive or veterinary druguse, a crude mixture of neoviridogriseins and griseoviridin will be moreadvantageous economically.

Neoviridogriseins and griseoviridin in the fermentation broth can beextracted with a water-immiscible organic solvent. For example, ethylacetate, butyl acetate, n-butanol, methylene chloride, chloroform, andthe like are suitable for extraction of neoviridogriseins andgriseoviridin at one time. When it is more desirable to selectivelyextract neoviridogriseins without griseoviridin, preferred organicsolvents are methyl isobutyl ketone, benzene, toluene and other aromatichydrocarbons. As the mycelium substantially contains noneoviridogriseins and the extractable lipid in cells may often interferewith subsequent purification steps, it is more profitable to extract thesaid antibiotics with an organic solvent from the filtered broth orcentrifuged broth together with the water wash.

The solvent extract of neoviridogriseins and/or griseoviridin can befurther isolated and purified in a number of different ways. Forexample, adsorption and elution processes with active carbon, AmberliteXAD-4 and 7 (Rohm & Haas Co.), ion exchange resins such as AmberliteIR-120 (Rohm and Haas Co.) and Dowex 50W-X2 (The Dow Chemical Co.); gelfiltration with Sephadex LH-20 (Pharmacia Fine Chemicals AB) and itsequivalents; adsorption chromatography on alumina and silica gel, etc.can be conveniently combined for isolation and purification. Inaddition, countercurrent distribution with a suitable solvent system maywork well for the said purposes.

PHYSICO-CHEMICAL PROPERTIES OF NEOVIRIDOGRISEINS I, II AND III

Neoviridogriseins I, II and III as well as viridogrisein are amorphouswhite solids and are soluble in methanol, ethanol, n-propanol,iso-propanol, n-butanol, dioxane, ethyl acetate, butyl acetate, acetone,methyl ethyl ketone, methyl isobutyl ketone, benzene, toluene, methylenechloride, chloroform, carbon tetrachloride, N,N-dimethylformamide anddimethyl sulfoxide; hardly soluble in water and ethyl ether; andpractically insoluble in petroleum ether and hexane.

They are stable in aqueous solution for at least one month at atemperature in the range of 25°-37° C.; and for 30 minutes at 60° C., ata pH of 2-9. The melting points of the antibiotics were determined in aKofler apparatus and the following results were obtained:

    ______________________________________                                        Neoviridogrisein I                                                                            not determined                                                II              93° C.                                                 III             115° C.                                                Viridogrisein   140° C.                                                ______________________________________                                    

The ultraviolet absorption spectra of neoviridogriseins I, II, III andviridogriseins are reproduced in FIGS. 1-8 wherein FIGS. 1-4 show theU.V. spectrum of neoviridogrisein I, II, III and viridogrisein whenregistered in methanol and FIGS. 5-8 the U.V. spectrum of the samesubstances registered in 0.1N NaOH-methanol. The E₁ cm.^(1%) value ofneoviridogrisein at their maxima is as follows:

In neutral methanol,

    ______________________________________                                        Neoviridogrisein I                                                                            305 nm (65)                                                   II              305 nm (88)                                                   III             305 nm (90)                                                   Viridogrisein   305 nm (90)                                                   ______________________________________                                    

In 0.1N NaOH-methanol,

    ______________________________________                                        Neoviridogrisein I                                                                            340 nm (70)                                                   II              340 nm (84)                                                   III             340 nm (96)                                                   Viridogrisein   340 nm (96)                                                   ______________________________________                                    

The infrared absorption spectra of neoviridogriseins I, II, III andviridogrisein, in a KBr tablet are shown in FIGS. 9-12 respectively. Thecharacteristic peaks and shoulders are observed at the following wavenumbers:

Neoviridogrisein I (KBr tablet): 3370, 2910, 2850, 1735, 1670(sh.),1635,1590(sh.),1515,1460(sh.)1445,1405,1375,1290,1280,1250(sh.),1200,1190,1160,1125,1100,and 1080 cm.⁻¹

Neoviridogrisein II(KBr tablet):3320,2950,2920,2820,2800,1745,1670(sh.),1630,1600(sh.),1575,1515,1460(sh.)1445,1405,1390,1365, 1330(sh.),1295,1275,1240,1200,1195,1160,1130,1095 and 1065 cm.⁻¹

Neoviridogrisein III(KBr tablet):3335,2960,2940,2870,1750,1670(sh.),1600(sh.),1635,1590(sh.);1515,1450,1405,1390(sh.),1370,1340(sh.),1300,1245,1200,1160(sh.),1130,1100 and 1065 cm.⁻¹

In the thin layer chromatography systems indicated below,neoviridogriseins I, II and III viridogrisein and griseoviridin have thefollowing Rf values:

    ______________________________________                                        (1) TLC plate                                                                             Pre-coated TLC plate SILICA GEL 60F-254,                                      E. Merck, Darmstadt.                                              Solvent     Benzene:methanol = 5:1                                            Neoviridogrisein I                                                                        Rf = 0.66                                                         II          0.62                                                              Neoviridogrisein III                                                                      Rf = 0.59                                                         Viridogrisein                                                                             0.55                                                              Griseoviridin                                                                             0.20                                                              (2) TLC plate                                                                             Same as (1)                                                       Solvent     Chloroform:methanol = 30:1                                        Neoviridogrisein I                                                                        Rf = 0.39                                                         II          0.32                                                              III         0.19                                                              Viridogrisein                                                                             0.18                                                              Griseoviridin                                                                             0.02                                                              ______________________________________                                    

For the analysis of the constituent amino acids, each neoviridogriseincomponent was hydrolysed in 6N HCl overnight at 110° C. and theresulting hydrolysate was evaporated to dryness. After even a trace ofhydrochloric acid was removed by repeated evaporations, amino acids inthe hydrolysate were determined by thin layer chromatography (EastmanChromagram sheet 13254 cellulose with fluorescent indicator, EastmanKodak Co.; solvent system:n-butanol/acetic acid/water=4/1/1), highvoltage paper electrophoresis (Toyo Filter paper No. 51A, Toyo RoshiKaisha, Ltd.; buffer system: formic acid/acetic acid/water=25/75/900,pH=1.8; 60 V/cm at 0° C. for 30 minutes) and auto-amino acid analysis(Hitachi auto-amino acid analyser KLA-5, Hitachi, Ltd.). The presence ofthe following amino acids was confirmed:

    ______________________________________                                        Neoviridogrisein I                                                                             threonine                                                                     leucine                                                                       proline                                                      Neoviridogrisein I                                                                             alpha-amino-n-butyric acid                                                    sarcosine                                                                     phenylsarcosine                                                               β, N--dimethylleucine                                   Neoviridogrisein II                                                                            threonine                                                                     leucine                                                                       proline                                                                       alanine                                                                       sarcosine                                                                     phenylsarcosine                                                               β, N--dimethylleucine                                   Neoviridogrisein III                                                                           threonine                                                                     leucine                                                                       hydroxyproline                                                                alpha-amino-n-butyric acid                                                    sarcosine                                                                     phenylsarcosine                                                               β, N--dimethylleucine                                   Viridogrisein    threonine                                                                     leucine                                                                       hydroxyproline                                                                alanine                                                                       sarcosine                                                                     phenylsarcosine                                                               β, N--dimethylleucine                                   ______________________________________                                    

The presence of 3-hydroxy-picolinic acid was confirmed by massspectrometry and thin layer chromatography as follows:

An authentic sample of viridogrisein and each of neoviridogriseins I,II, III and IV were hydrolysed overnight in 6N HCl at 110° C. to givethe hydrolysates as described above. Each hydrolysate showed only oneUV-absorbing spot with the same Rf value under the indicated conditions.

(1) Silica gel TLC

TLC plate: Pre-coated TLC plate SILICA GEL 60 F-254, E. Merck,Darmstadt.

Solvent: Chloroform:methanol=2:1

Rf: 0.46

(2) Cellulose TLC

TLC plate: Eastman Chromagram sheet 13254 cellulose with fluorescentindicator, Eastman KodaK Co.

Solvent: n-Butanol:acetic acid:water=4:1:1

Rf: 0.62

The molecular weight of these antibiotics was determined by directinsertion into a mass spectrometer.

    ______________________________________                                        Neoviridogrisein I                                                                             876                                                          II               862                                                          III              892                                                          Viridogrisein    878                                                          ______________________________________                                    

For the study on the chemical structure of neoviridogriseins I, II andIII, these three new antibiotics and viridogrisein were hydrolysedovernight in 0.1N NaOH at room temperature and then methylated withdiazomethane before mass spectrometry according to the method ofCompernolle et al.(Organic Mass Spectrometry, Vol. 6, pp151-166, 1972).The structure of neoviridogriseins I, II and III is concluded from theavailable information described above to be as follows: ##STR1##

As can be seen from the preceding structures of neoviridogriseins I, IIand III, the antibiotics of the invention are a group of newdepsipeptide antibiotics homologous to viridogrisein. The identity ofneoviridogrisein IV with viridogrisein and etamycin was confirmed bymass spectrometry, thin layer chromatography, UV and IR spectrometry andamino acid analysis, using authentic samples of viridogrisein andetamycin.

ANTIMICROBIAL ACTIVITY

Neoviridogrisein I, II and III have a broad antimicrobial spectrumagainst bacteria, mycoplasmas, actinomycetes and rickettsiae inlaboratory test. More exactly, they display a remarkable activity invitro against the usual and resistant strains of Staphylococcus aureus,as well as strains of Streptococcus pyogenes, Diplococcus pneumoniae,Sarcina lutea, Bacillus subtilis, mycoplasma gallisepticum, Mycoplasmapulmonis, Mycoplasma fermentans and Mycoplasma agalactiae. The minimalinhibitory concentrations of the new depsipeptide antibiotics of thisinvention were determined separately and together with viridogrisein andgriseoviridin on various microorganisms by the tube dilution method. Theresults are shown in the following tables:

                                      TABLE 1                                     __________________________________________________________________________    MIC values of neoviridogriseins I, II and III                                 (mcg/ml)                                                                      Microorganism (a)                                                                            Medium NV*I NV*II                                                                              NV*III                                                                             VG** NV* mix.                                                                           NV* + GV***                    __________________________________________________________________________                                                   mix.                           Staphylococcus aureus                                                         209P           BHI.sup.#                                                                            0.2  0.1  0.2  0.2  0.2  0.1                            (EM).sup.r     BHI.sup.#                                                                            0.4  0.4  0.4  0.4  0.4  0.2                            (STH).sup.r    BHI.sup.#                                                                            0.8  0.8  1.6  1.6  1.6  0.8                            (PC, TC, EM, LM).sup.r 1                                                                     BHI.sup.#                                                                            6.25 6.25 6.25 6.25 6.25 3.2                            (PC, TC, EM, LM).sup.r 2                                                                     BHI.sup.#                                                                            6.25 6.25 6.25 6.25 6.25 3.2                            (TC, EM, LM, CP).sup.r                                                                       BHI.sup.#                                                                            6.25 6.25 6.25 6.25 6.25 3.2                            (SM, STH).sup.r                                                                              BHI.sup.#                                                                            0.4  0.2  0.2  0.4  0.4  0.2                            (PC, EM, NM).sup.r                                                                           BHI.sup.#                                                                            0.8  0.4  0.4  0.8  0.8  0.4                            (EM, CM, SM, PC, TC).sup.r                                                                   BHI.sup.#                                                                            0.4  0.2  0.4  0.4  0.4  0.2                            (EM, OM).sup.r BHI.sup.#                                                                            0.8  0.4  0.8  0.8  0.8  0.4                            (TC, CP, PC).sup.r                                                                           BHI.sup.#                                                                            0.2  0.2  0.4  0.4  0.4  0.2                            (BX-1633)(PC).sup.r                                                                          BHI.sup. #                                                                           0.4  0.2  0.4  0.4  0.4  0.2                            Russell(PC).sup.r                                                                            BHI.sup.#                                                                            0.2  0.2  0.4  0.4  0.4  0.2                            Smith          BHI.sup.#                                                                            0.2  0.2  0.2  0.4  0.4  0.2                            Staphylococcus sp. (PC).sup.r                                                                BHI.sup.#                                                                            0.8  0.4  0.8  0.4  0.8  0.4                            Staphylococcus sp.                                                                           BHI.sup.#                                                                            0.8  0.8  0.8  0.8  0.8  0.4                            Diplococcus pneumoniac type 1                                                                BHI + HB.sup.                                                                        0.2  0.4  0.2  0.4  0.4  0.1                            Streptococcus pyogenes                                                                       BHI + HB.sup.                                                                        0.4  0.2  0.4  0.4  0.4  0.1                            Saccina lutea  BHI.sup.#                                                                            0.2  0.4  0.4  0.2  0.4  0.1                            Bacillus subtilis ATCC 6633                                                                  BHI.sup.#                                                                            0.4  0.4  0.8  0.8  0.8  0.4                            Salmonella gallinanum                                                                        BHI.sup.#                                                                            >25  >25  >25  >25  >25  >25                            Escherichia coli                                                                             BHI.sup.#                                                                            >25  >25  >25  >25  >25  >25                            Pseudeomonas aeruginosa                                                                      BHI.sup.#                                                                            >25  >25  >25  >25  >25  >25                            Proteus vulgaris                                                                             BHI.sup.#                                                                            >25  >25  >25  >25  >25  >25                            Candida albicans                                                                             MY.sup.&                                                                             >25  >25  >25  >25  >25  >25                            __________________________________________________________________________     *NV = neoviridogrisein;                                                       **VG = viridogrisein;                                                         ***GV = griseoviridin                                                         (a)EM = erythromycin;                                                         STH = streptothricin;                                                         PC = penicillin;                                                              TC = tetracycline;                                                            LM = leucomycin;                                                              CP = chloramphenicol;                                                         SM = streptomycin;                                                            KM = kanamycin;                                                               NM = neomycin;                                                                OM = oleandomycin                                                             ().sup.r = resistant to the drug(s) in parenthesis                            .sup.# BHI =  brain heart infusion broth;                                     .sup.  BHI + HB = brain heart infusion broth containing 10% horse blood;      .sup. & MY = malt extractyeast extract medium                            

As shown in the table of MIC values listed above, neoviridogrisein II ismore active than neoviridogrisein IV, that is, viridogrisein. This MICexperiment was based on the two-fold dilutions. To differentiateneoviridogrisein II and viridogrisein in their antibiotic activity, theMIC determination was repeated with a far lower dilution rate. Thesubsequent table indicates that neoviridogrisein II is 2-3 times moreactive than viridogrisein.

                  TABLE 2                                                         ______________________________________                                        Comparison of neoviridogrisein II with viridogrisein                          Microoganism                                                                                     MIC(mcg/ml)                                                                   NV*II VG**                                                 ______________________________________                                         Staphylococcus aureus 209 P                                                                       0.078   0.133                                            (EM, CM, SM, PC, TC).sup.r                                                                         0.125   0.334                                            (TC, CP, PC).sup.r   0.094   0.334                                            Bx-1633(PC).sup.r    0.125   0.267                                            Russell(PC).sup.r    0.125   0.267                                            Smith                0.125   0.267                                            ______________________________________                                         Medium: brain heart infusion broth                                            Abbreviation: as listed above.                                           

When the minimal inhibitory concentrations of neoviridogriseins I, IIand III were tested in the presence of griseoviridin, the synergism wasobserved between the neoviridogrisein member and griseoviridin, as isthe case of viridogrisein and griseoviridin. Therefore the synergisticphenomenon of the new antibiotics of this invention with griseoviridinwas studied in more detail with the varied ratios of theneoviridogriseins mixture to griseoviridin. The obtained results areshown in the following table:

                  TABLE 3                                                         ______________________________________                                        Synergism of neoviridogriseins mixture with griseoviridin                     Neoviridogriseins                                                             mixture       Griseoviridin                                                                            MIC* (mcg/ml)                                        ______________________________________                                        100            0         0.313                                                90            10         0.156                                                80            20         0.125                                                70            30         0.125                                                60            40         0.094                                                50            50         0.078                                                40            60         0.094                                                30            70         0.125                                                20            80         0.250                                                10            90         0.250                                                 0            100        0.250                                                ______________________________________                                         *Test microorganism Sarcina lutea                                        

Tube dilution method with brain heart infusion broth. The above tableindicates that the synergistic action of the neoviridogriseins mixturewith griseoviridin was most significant at the ratio of 50:50; that is,a 1:1 mixture of neoviridogriseins and griseoviridin is 3-4 times moreactive than neoviridogriseins or griseoviridin only.

The following table 4 reports the high in vitro activity ofneoviridogrisein II against various Mycoplasma strains as well as thesuperior synergism shown by a mixture neoviridogrisein II-griseoviridinin comparison with a mixture viridogrisein-griseoviridin. The MIC weredetermined by the dilution method.

                                      TABLE 4                                     __________________________________________________________________________    Microorganism Medium                                                                             NV II                                                                             VG  GV  NV II + GV*                                                                          VG + GV*                                __________________________________________________________________________    Mycoplasma gallisepticum                                                                    (1)  0.025                                                                             0.10                                                                              0.10                                                                              0.0063 0.025                                   KP13                                                                          Mycoplasma pulmonis PG 22                                                                   (2)  0.78                                                                              1.56                                                                              6.25                                                                              0.20   0.39                                    Mycoplasma fermentans                                                                       (2)  <0.05                                                                             <0.05                                                                             <0.05                                                                             <0.05  <0.05                                   Mycoplasma agalactiae PG 2                                                                  (2)  0.39                                                                              0.78                                                                              3.13                                                                              <0.05  0.39                                    __________________________________________________________________________     Medium (1): PPLO enrichment broth (Eiken, Japan)                              (2): PPLO broth (Chanock's medium; Difco)                                     *Mixing ratio: 50/50                                                          NV II, VG, GV: see table 1                                               

As illustrated above, the novel neoviridogriseins I-III of the presentinvention display a remarkable activity against gram-positive bacteriaand mycoplasma strains both alone or in various admixtures withviridogrisein (neoviridogrisein IV) and griseoviridin. It has been foundthat the mutual weight proportions of neoviridogriseins I-III,viridogrisein (neoviridogrisein IV) and griseoviridin in a mixture mayvary within very wide limits, but the surprising antimicrobial andanti-mycoplasma activities are still retained. As a representative,though not limitative example, a mixture having the following percentcomposition (by weight):

Neoviridogrisein II: 27%

Neoviridogrisein IV(viridogrisein): 23%

Griseoviridin: 50%

was tested in vitro against Streptococcus mutans, a microorganismassociated with dental caries and peridontal diseases. The test wascarried out in Todd Hewitt Broth (Difco) with 0.5% TC Lactalbuminhydrolysate (Difco). Initial organism count approximately 3×10⁴organisms per ml. Culture tubes were incubated anaerobically at 37° C.for 48 hours. The minimum inhibitory concentration (MIC) and minimumbactericidal concentration were both found at a neoviridogriseinconcentration of 1.0 part per million (ppm).

In another representative, though not limitative example, the samemixture was tested in vitro against Treponema hyodysenteriae, a swinedysentery organism. The mixture was tested as dilutions in blood agar atconcentrations of 100, 50, 10, 5, 1, 0.5 and 0.1 ppm. Plates wereinoculated with a swab and incubated 4 days at 42° C. The MIC wasdetermined to be 0.5 ppm. In a further representative, though notlimitative example, a mixture having the following percent composition(by weight):

Neoviridogrisein II: 25%

Neoviridogrisein IV (viridogrisein): 25%

Griseoviridin: 50%

was tested in vitro agains several Mycoplasma strains.

The minimum inhibitory concentrations (MIC) were found to be as follows:

    ______________________________________                                        Strain               MIC                                                      ______________________________________                                        Mycoplasma gallisepticum KP13                                                                      0.07                                                     Mycoplasma pulmonis  0.20                                                     Mycoplasma fermentans                                                                              0.05                                                     Mycoplasma agalactiae                                                                              0.05                                                     ______________________________________                                    

These types of neoviridogriseins-griseoviridin mixtures are also usefulin the treatment of animals suffering from infections diseases caused bythe above pathogenic bacteria.

As mikamycins A and B have been known to be very effective as feedadditive, the new antibiotic compounds of this invention were subjectedto the animal feed test. Neoviridogriseins as mixture were added to thechicken feed at a rate of 2-20 ppm and fed to male chickens for 10weeks. Compared with the control group of chicken which received thesame feed without neoviridogriseins, the neoviridogrisein-fed chickenswere superior in the increase rate of body weight and the feedefficiency. Thus neoviridogriseins of this invention have been provedvery useful as feed additive.

Also the compositions containing one or more of the neoviridogriseinsI-III, optionally in admixture with viridogrisein (neoviridogrisein IV)and griseoviridin, wherein the mutual weight proportions of thecomponents may vary within very wide limits, proved to be very useful asfeed additive.

As a representative, though not limitative example, a mixture having thefollowing percent composition (by weight):

Neoviridogrisein II: 27%

Neoviridogrisein IV (viridogrisein): 23%

Griseoviridin: 50%

was tested in vivo by incorporation in the diet of growing chicks.One-day old cockerels, 15 birds per treatment were used. The chicks weremaintained for 11 days on a feed containing about 55% rye grain,supplemented with vitamins, minerals, fat and protein sources. The highcontent of rye grain normally gives poor to moderate growth, and thisdiet is a standard one used for screening growth promoters andantibiotic feed additives. Penicillin (100 ppm) was used as a positivecontrol. The data is tabulated below. Feed/Gain ratio is grams feedconsumed per gram of weight gained. Body weight ratio is the ratio ofchick body weight at the end of the 11 day study to the initial bodyweight. The last column is the average gain per bird (in grams).

    ______________________________________                                                  DIET    FEED/     BODY    (GMS)                                               CONC    GAIN      WEIGHT  AVG                                       TREATMENT PPM     RATIO     RATIO   B.W.GAIN                                  ______________________________________                                        Rye Control                                                                             --      1.334     4.696   156.47                                    Penicillin                                                                              100     1.197     5.012   163.20                                    Tested    100     1.256     4.789   161.66                                    mixture                                                                       Tested     50     1.283     4.892   163.47                                    mixture                                                                       Tested     25     1.204     5.226   174.67                                    mixture                                                                       ______________________________________                                    

In the following, this invention will be further illustrated bypreferred examples, but should not be construed as limited by thoseexamples.

EXAMPLE 1

Seed culture medium consisting of soybean meal 0.5%, Pharmamedia(Traders Oil Mill Co.) 0.5%, oatmeal 0.5%, dry yeast 0.5% and beetmolasses 0.5% was adjusted to pH 6.5 and distributed in a 50 ml. amountin a 250 Erlenmeyer flask. After autoclaving at 120° C. for 15 minutes,a loopful amount of Streptomyces sp. P 8648 on an ISP-2 agar slant wasinoculated and the flask incubated at 28° C. for 48 hours on a rotaryshaker. Two milliliters of the said seed culture was transferred into a500 ml. Erlenmeyer flask containing 100 ml. of the followingfermentation medium:

Soybean meal: 0.5%

Peanut meal: 0.5%

Oatmeal: 0.5% (pH 6.5 prior to autoclaving)

Dry yeast: 0.5%

Beet molasses: 0.5%

and cultivated at 28° C. for 96 hours on a rotary shaker at 200 r.p.m.(radius of circle 3.5 cm.). The culture broth was collected from 12flasks and filtered to give a clear broth filtrate. The obtained cake onthe filter was washed with 100 ml. of water. The water washing and thebroth filtrate were combined. The antibiotic activity of this solution(pH 8.3) was determined to be 23.0 mm on a nutrient agar assay plate ofSarcina lutea when the standardized disc assay was carried out with a 8mm paper disc. Eight hundred milliliter of the said aqueous solution wastwice extracted with 200 ml. each of n-butanol, and the butanol extractswere combined and evaporated to dryness under reduced pressure to yield90 mg. of crude powder of neoviridogriseins and griseoviridin. Thiscrude powder was mixed with a small amount of silica gel and applied ona silica gel column (Wako-Gel C-100, Wako Pure Chemical Industries,Ltd.; 1.5×25 cm.). The silica gel column was eluted first with 300 ml.of a benzene-acetone mixture (5:1), and then with a benzene-acetonemixture (2:1). Ten gram fractions were collected on an automaticfraction collector. Active fractions of Nos. 25 to 35 were combined andevaporated to dryness to provide 30 mg. of neoviridogrisein mixture(consisted of neoviridogriseins I, II and III and viridogrisein). Inaddition, evaporation of active fractions of Nos. 45-54 to dryness gavea crude powder, the antibiotic activity of which corresponded togriseoviridin by TLC. These two preparations were subjected to thinlayer chromatography under the indicated conditions. The antimicrobialactivity was detected on a nutrient agar assay plate of Sarcina lutea.

TLC plate: Pre-coated TLC plate SILICA GEL 60 F-254, E. Merck,Darmstadt.

(1) Solvent: Chloroform:methanol=20:1

Neoviridogriseins Rf=0.45

Griseoviridin: 0.05

(2) Solvent: Benzene:acetone=1:1

Neoviridogriseins Rf=0.55

Griseoviridin: 0.13

EXAMPLE 2

Two hundred milliliters of the 48 hour-old culture of Streptomyces sp.P8648 in the same culture medium as in Example 1 was inoculated into a15 liter stainless steel jar fermenter containing 10 liters of the sameseed culture medium as in Example 1 and cultivated at 27°-28° C. for 96hours under forced aeration of 5 liters/min. of sterile air. Theagitation during cultivation was performed at 200 r.p.m. with animpeller, the radius of which is about a fourth of the diameter of thejar fermenter. At the end of fermentation, the mycelia and solids wereremoved by filtration. The obtained broth filtrate was adjusted to pH6.0 and extracted four times with 2 liters each of ethyl acetate. Theethyl acetate extracts were combined, dried over anhydrous sodiumsulfate and evaporated to dryness under reduced pressure to yield 700mg. of crude neoviridogriseins and griseoviridin. The recovered crudepowder of neoviridogriseins and griseoviridin was dissolved in a smallamount of methanol and charged on a Sephadex LH-20 column (3×50 cm.).Ten milliliter fractions were collected with methanol as an elutingsolvent. Neoviridogriseins were located in fraction Nos. 22-29. Thesefractions were collected, concentrated to dryness and further purifiedby silica gel column chromatography (SILICAR CC-7 Special; MallinckrodtChemical Works; 1.5×20 cm.). The elution was run with a mixture ofchloroform and methanol (30:1). Eight 5 gram fractions from fraction No.5 to fraction No. 12 were combined and evaporated to dryness underreduced pressure to yield 25 mg. of white powder of neoviridogriseins.The percent composition of neoviridogriseins I, II and III andviridogrisein in this powder was as follows:

    ______________________________________                                        Neoviridogrisein I                                                                             15%                                                          II               20%                                                          III              20%                                                          Viridogrisein    45%                                                          ______________________________________                                    

Griseoviridin was found by TLC in fraction Nos. 30-34 of the saidSephadex LH-20. These active fractions were combined, evaporated todryness under reduced pressure and crystallized in warm methanol to give30 mg. of needle crystals of griseoviridin. The identity of thesecrystals with griseoviridin was proved by TLC and other physico-chemicaldeterminations.

EXAMPLE 3

The same fermentation as described in Example 1 was carried out for 96hours except that the fermentation medium was composed of soybean meal0.5%, Pharmamedia 0.5%, oatmeal 0.5%, dry yeast 0.5%, beet molasses 0.5%and DL-alpha-amino-n-butyric acid 0.1% (pH 6.5). The fermentation brothwas collected from 13 flasks and filtered. The filtered liquid wasextracted with n-butanol (300 ml. each, twice). The removal of n-butanolfrom the extracts left about 70 mg. of crude powder of neoviridogriseinsand griseoviridin. This crude powder was analysed by silica gel thinlayer chromatography followed by bio-autography on Sarcina lutea as thetest organism. The TLC plate employed in this assay was a product of E.Merck, Darmstadt (Pre-coated TLC plate SILICA GEL 60 F-254). Theobtained Rf values and the solvent systems were as follows:

    ______________________________________                                        Chloroform:methanol                                                                          20:1          30:1   40:1                                      ______________________________________                                        Neoviridogrisein I                                                                           Rf = 0.60     0.39   0.20                                      II             0.56          0.32   0.16                                      III            0.50          0.19   0.13                                      Viridogrisein  0.43          0.18   0.10                                      Griseoviridin  0.05          0.02   0.00                                      ______________________________________                                    

EXAMPLE 4

Fifty milliliters of the seed culture medium containing 0.3% beefextract, 0.5% tryptone (Difco laboratories), 0.1% glucose, 2.4% solublestarch, 0.5% yeast extract, 0.4% calcium carbonate and 0.5% soybean meal(pH 7.0) was distributed in a 250 ml. Erlenmeyer flask and autoclaved at120° C. for 15 minutes. Spores of Streptomyces sp. P 8648 on a agarslant were seeded in the said flask and shake-cultured at 25° C. for 3days to supply the seed culture. The fermentation medium was composed of0.5% soluble starch, 2.0% glucose, 1.0% Pharmamedia, 0.5% oatmeal, 0.5%corn steep liquor, 0.05% dipotassium phosphate and 0.05% magnesiumsulfate (pH 6.5). Fifty milliliters of this fermentation medium wasplaced in a 250 ml. conical flask and autoclaved at 120° C. for 15minutes. The size of the inoculum was 2% (v/v). The fermentation flaskwas inoculated with the above described seed culture and incubated at25° C. on a rotary shaker. Twenty four or 48 hours after inoculation, asterilized solution of casamino acid (Difco Laboratories) or proline (pH7.0) was added to a final concentration of 0.4% and the fermentation wascontinued. Four days after inoculation, the fermentation broth wasfiltered and the broth filtrate was extracted twice with an equal volumeof ethyl acetate. The ethyl acetate extracts were combined andevaporated to dry powder under reduced pressure. This crude powder wastaken in ethyl acetate and an aliquot amount of the solution wasquantitatively spotted on a silica gel TLC plate. The TLC plate wasdeveloped in a solvent system of chloroform and methanol (100:1) and thesolvent was evaporated off in the air. The same TLC plate was againdeveloped in the said solvent system of chloroform and methanol (100:1).Each component of neoviridogriseins was located under ultra-violet light(3650 Å), scraped off from the TLC plate and suspended in a known volumeof methanol. After the silica gel was removed by decantation, the amountof the antibiotics in the extracts was determined by the UV-assaymethod, knowing that the epsilon value at 305 nm is 8,000. The resultsof the UV-assay are as follows:

    ______________________________________                                                   Amino acid added                                                              None    Casamino acid                                                                             Proline                                        ______________________________________                                        Neoviridogrisein I                                                                         2%        2%          2%                                         II           9         40          60                                         III          4         3           3                                          Viridogrisein                                                                              85        55          35                                         ______________________________________                                    

EXAMPLE 5

About 10 liters of the 23 hour-old seed culture was prepared in a jarfermenter under the same condition as described in Example 2. Thefermentation medium (600 liter) comprising 0.5% soybean meal, 0.5%Pharmamedia, 0.5% oatmeal, 0.5% dry yeast, 0.5% beet molasses and 0.1%DL-alpha-amino-n-butyric acid (pH adjusted to 6.5 prior to autoclaving)was steam-sterilized at 120° C. for 15 minutes in a 1400 liter stainlesssteel tank fermenter and cooled down to 28° C. To this tank fermenter,the above-mentioned seed culture (10 liters) was added and cultivated at28° C. for 75 hours under forced aeration with stirring at 180 r.p.m.(by means of a double impeller; radius of circle, 1/4 of the diameter ofthe tank fermenter), the sterile air being fed at 300 liters/minutethrough a sparger from the bottom of the tank. At the end offermentation, the broth was filtered through a filter press. The brothfiltrate was extracted twice with 150 liters each of n-butanol. Then-butanol extracts were combined, washed with a small volume ofsaturated NaCl solution, and concentrated to 2 liters in a rotaryevaporator. At this stage, 20 grams of silica gel (WAKOGEL G-100, WakoPure Chemical Industries, Ltd.) was added and the concentration in arotary evaporator was further continued to complete dryness. Theobtained antibiotics-silica gel mixture was suspended in a small amountof chloroform and put on top of a silica gel column (WAKOGEL C-100;6.5×75 cm.). The elution of the antibiotic activities was carried outstepwise first with 7 liters of chloroform, then with 10 liters of amixture of chloroform and methanol (50:1); and finally with methanol.Fraction Nos. 16-29 (300 g/fraction) which were found bio-active onSarcina lutea (neoviridogriseins) were collected and concentrated todryness under reduced pressure to yield a crude powder ofneoviridogriseins. This crude powder was dissolved in a small volume ofmethanol and passed through a Sephadex LH-20 column (7.0×45 cm.), eachfraction (100 g.) being eluted with methanol. About 15 grams of crudepowder of neoviridogrisein mixture was recovered from fraction Nos. 6-15after the solvent was removed by evaporation. Fraction Nos. 50-60 of theabove described silica gel column contained griseoviridin. A similarpurification procedure with Sephadex LH-20 (column size 7.0×45 cm.) asused for the neoviridogrisein mixture was repeated to provide 4 grams ofcrude griseoviridin.

EXAMPLE 6

For final purification, preparative thin layer chromatography with asilica gel TLC plate was employed. One gram of the crudeneoviridogrisein mixture prepared in Example 5 was dissolved in 2 ml. ofethyl acetate and applied in bandwise fashion on ten silica gel TLCplates (Pre-coated TLC plate SILICA GEL 60 F-254). These TLC plates werefirst developed with a solvent system of chloroform and methanol (50:1).After the solvent was evaporated off in the air, the said TLC plateswere subjected to a second development with a solvent system ofchloroform and methanol (25:1). Neoviridogriseins I, II and III andviridogrisein were marked on the TLC plates under ultra-violet light(3650 A; BLAK-RAY UVL-22, Ultra-Violet Products, Inc.) and scraped offthe elution. Each neoviridogrisein component was eluted with a smallamount of methanol and evaporated to dryness. The recovered amount ofeach neoviridogrisein component in pure state was as follows:

    ______________________________________                                        Neoviridogrisein I                                                                          16.7 mg. (less pure, oily)                                      II            11.1 mg. (white powder)                                         III           15.2 mg. (white powder)                                         Viridogrisein 30.0 mg. (white powder)                                         ______________________________________                                    

About 5 mg. each of the neoviridogriseins was hydrolyzed in 6N HCl at110° C. for 36 hours in a sealed tube and subjected to thin layerchromatography, paper chromatography, high voltage paper electrophoresisand autoamino acid analysis. The presence of the following constituentcompounds was found in each component:

    ______________________________________                                        Neoviridogrisein I                                                                              3-hydroxy-picolinic acid                                                      threonine                                                                     leucine                                                                       proline                                                                       sarcosine                                                                     β,N--dimethylleucine                                                     α-amino-n-butyric acid                                                  phenylsarcosine                                             Neoviridogrisein II                                                                             3-hydroxypicolinic acid                                     Neoviridogrisein II                                                                             threonine                                                                     leucine                                                                       proline                                                                       sarcosine                                                                     β,N--dimethylleucine                                                     alanine                                                                       phenylsarcosine                                             Neoviridogrisein III                                                                            3-hydroxy-picolinic acid                                                      threonine                                                                     leucine                                                                       hydroxyproline                                                                sarcosine                                                                     β,N--dimethylleucine                                                     alpha-amino-n-butyric acid                                                    phenylsarcosine                                             Viridogrisein     3-hydroxypicolinic acid                                                       threonine                                                                     leucine                                                                       hydroxyproline                                                                sarcosine                                                                     β,N--dimethylleucine                                                     alanine                                                                       phenylsarcosine                                             ______________________________________                                    

In addition the identity of neoviridogrisein IV with viridogrisein wasfurther confirmed by IR, UV, NMR and mass spectrometly, thin layerchromatography, hydrolysate analysis and antimicrobial spectrometry. Onthe other hand, the griseoviridin preparation obtained in Example 5 wascrystallized in warm methanol to yield needle crystals. Then a part ofthe needle crystals were compared and identified with an authenticpreparation of griseoviridin by IR, UV, NMR and mass spectrometry, thinlayer chromatography, elementary analysis and other physico-chemicalproperties.

We claim:
 1. A process for preparing depsipeptide antibioticneoviridogrisein I of the formula ##STR2## depsipeptide antibioticneoviridogrisein II of the formula ##STR3## depsipeptide antibioticneoviridogrisein III of the formula ##STR4## which comprises cultivatingStreptomyces sp. P 8648 (FERM-P3562; ATCC 31289) under aerobicconditions, at a temperature between 18° and 37° C., in an aqueousnutrient medium containing assimilable sources of carbon, assimilablesources of nitrogen, and essential mineral salts, at a pH between about6 and about 9, until a substantial antibiotic activity is imparted tothe medium, recovering the fermentation product from the medium and,optionally, isolating neoviridogrisein I, neoviridogrisein II andneoviridogrisein III as the single components.
 2. The method of claim 1,wherein, in addition to neoviridogrisein I, neoviridogrisein II andneoviridogrisein III, viridogrisein and griseoviridin are coproduced. 3.The method of claim 1, wherein the fermentation is carried out in thepresence of alpha-amino-n-butyric acid and/or natural sources containingalpha-amino-n-butyric acid.
 4. The method of claim 1 wherein thefermentation is carried out in the presence of proline and/or a naturalsource containing proline.
 5. An antibiotic composition produced byStreptomyces sp. P 8648 (FERM-P3562; ATCC 31289) consisting ofneoviridogrisein I, neoviridogrisein II, neoviridogrisein III,viridogrisein and griseoviridin.